Project Summary The human fetus depends on the placenta for the acquisition of nutrients including lipids. The long-chain polyunsaturated fatty acids are crucial building blocks for the developing nervous and cardiovascular systems. It is thought that the placenta selectively incorporates long-chain polyunsaturated fatty acids into an esterified lipid pool before being released to the fetus as free-fatty acids. However, the involvement of placental cell layers and mechanisms underlying placental fatty acid metabolism are unclear. Our preliminary work using a fluorescent long-chain fatty acid analogue, BODIPY-C12, suggests that the cytotrophoblast that underlies the maternal facing syncytiotrophoblast is responsible for placental long-chain fatty acid uptake and incorporation into esterified lipids. The cytotrophoblast layer was not known to play a direct-role in placental fatty acid esterification. Here, we propose to test the hypothesis that the cytotrophoblast cell is responsible for selective incorporation of long-chain polyunsaturated vs. saturated fatty acids into esterified lipids within the placenta using lipidomic technology. Aim 1: To identify the lipid species produced in isolated cytotrophoblasts, using lipidomics, before and after syncytialization after incubation with an unsaturated essential omega-3 fatty acid (Docosahexaenoic acid, DHA) compared to saturated fatty acid (Palmitic acid, PA). Aim 2: To determine the subcellular localization and chromatographic profile in explanted term villous placentas and cultured cytotrophoblasts fed a BODIPY-tagged saturated long-chain fatty acid analogue (BODIPY-C12) and a BODIPY- tagged custom synthesized unsaturated fatty acid analogue (BODIPY- C12X) of similar size. Lipidomic profiles from placental tissue incubated with heavy-labeled DHA will be compared to those lipid profiles of isolated cytotrophoblast incubated under the same conditions. The syncytiotrophoblast, the cell layer previously believed to selectively esterify DHA in the placenta will also be analyzed using these methods. Sex dependent comparisons will allow the identification of placental DHA-lipid products that are produced more highly in cytotrophoblast vs syncytiotrophoblast. This project seeks to define (1) the trophoblast layer responsible for a particular lipid milieu (metabolic products) when fed polyunsaturated or saturated long-chain fatty acids (2) the subcellular localization of saturated vs unsaturated fatty acids in syncytiotrophoblast and cytotrophoblast.